Binder composition and analytical element having stabilized peroxidase in layer containing the composition

ABSTRACT

An analytical element has a peroxidase-labeled ligand analog distributed within a water-soluble binder composition comprising at least about 50 percent, by weight, of poly(vinyl alcohol). As a result, the peroxidase retains more of its stability prior to use. Such elements can be used to determine any of a number of immunologically reactive analytes, such as digoxin.

FIELD OF THE INVENTION

The present invention relates to clinical chemistry. In particular, itrelates to binder compositions, to analytical elements containingstabilized peroxidase-labeled ligand analogs and to their use inanalytical methods to assay liquids, e.g. biological fluids.

BACKGROUND OF THE INVENTION

It is well known to perform a quantitative or qualitative analysis of anaqueous liquid by contacting that liquid with an analytical elementcontaining a combination of reagents capable of yielding a detectableproduct in proportion to the concentration of the predetermined analytein the liquid. As used herein, this combination of reagents is termed aninteractive composition which is capable of chemical reactivity,catalytic activity, or any other form of chemical or physicalinteraction that can result in the ultimate production of a change inthe element that is detectable with suitable procedures and equipment.

One type of particularly useful analytical elements utilizes enzymaticreactions wherein the analyte, upon contact with reagents in theelement, reacts with oxygen in the presence of a suitable enzyme toproduce peroxide in proportion to the concentration of the analyte. Adetectable product is then produced by the reaction of the peroxide inproportion to the concentration of the analyte in the tested liquid.Such useful elements are described, for example, in U.S. Pat. No.3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al).

Unfortunately, because of the intrinsic instabilities of certainreagents and the need to dry these down in contact with other materials,the reagents contained therein may deteriorate during storage and thusdeleteriously affect the accuracy and reliability of the assay. Forexample, exposure to air and moisture may adversely affect peroxidasewhich is often included in an element to catalyze the oxidation ofinteractive compositions by a peroxide.

Peroxidases are used for diverse purposes, including diagnosticdeterminations of analytes such as glucose, uric acid, cholesterol, etc.In such determinations, excess peroxidase can be added to an element toovercome the effect of enzyme deterioration during storage. However,enzyme immunoassays using a peroxidase-labeled ligand analog have becomeimportant for determining an immunologically reactive ligand such as adrug, antigen or other immunologically reactive compound. In suchassays, excess peroxidase cannot be added and the adverse effect of itsinstability is more prominent because of the relatively lowconcentration of ligand to be determined.

U.S. Pat. No. 4,283,491 (issued Aug. 11, 1981 to Dappen) describes thestabilization of peroxidase in elements with a vinyl copolymer preparedfrom specific ethylenically unsaturated polymerizable monomers. Theseelements further contain reagents needed for the determination ofparticular analytes such as glucose and uric acid. Immunoassays are notdescribed. The copolymer is included in the element carrier materials inan amount of from about 20 to about 50 weight percent. The remainder ofthe carrier materials can be one or more of a variety of bindermaterials, e.g. gelatin, hydrophilic celluloses, poly(vinyl alcohol),polysaccharides, etc.

It has been found, however, that peroxidase is not sufficientlystabilized by the materials described in U.S. Pat. No. 4,283,491 whenperoxidase is used as a label in a ligand analog. The problem ofperoxidase instability is more acute in the determination of a low levelligand in an immunoassay than in an assay of analytes such as glucose oruric acid which are generally found in test liquids in higherconcentrations. Therefore, there is a need for a means to stabilizeperoxidase-labeled ligand analogs in dry immunoassays.

SUMMARY OF THE INVENTION

The problems noted above are overcome with a water-soluble compositioncomprising a peroxidase-labeled ligand analog. The composition alsocomprises a water-soluble binder composition composed of at least about50 percent, by weight, of poly(vinyl alcohol) and, optionally one ormore additional binder materials.

This invention also provides an analytical element comprising anabsorbent carrier material containing a peroxidase-labeled ligand analogfor an immunologically reactive ligand uniformly distributed in thewater-soluble binder composition described above.

In a preferred embodiment, a multilayer analytical element comprises anonporous support having thereon, in order,

a registration layer,

a water-soluble layer containing a peroxidase-labeled ligand analog foran immunologically reactive ligand uniformly distributed in thewater-soluble binder composition described above, and

a porous spreading layer,

the element further comprising an interactive composition which iscapable of interacting with the ligand analog to provide aspectrophotometric signal in the presence of a substrate for peroxidase.

This invention also provides a method for the determination of animmunologically reactive ligand comprising the steps of:

A. in the presence of a receptor for an immunologically reactive ligand,contacting a sample of a liquid suspected of containing the ligand withan analytical element comprising an absorbent carrier materialcontaining a peroxidase-labeled ligand analog for the ligand uniformlydistributed in the water-soluble binder composition described above, thecontacting carried out in such a manner as to form a complex of receptorand ligand analog, and

B. determining the amount of the ligand as a result of the presence ofcomplexed or uncomplexed ligand analog.

The present invention provides a means for stabilizingperoxidase-labeled ligand analogs in analytical elements. The peroxidaseis stabilized sufficiently such that it can be retained in elements inlow concentrations. Therefore, analytes, e.g. immunologically reactiveligands present in a test fluid in low concentrations can be rapidly andaccurately determined. These advantages are achieved by putting theligand analog in a water-soluble binder composition which includes atleast about 50 percent, by weight, of poly(vinyl alcohol).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the determination (qualitative orquantitative measurement) of an immunologically reactive ligand inaqueous liquids. In particular, the invention can be used to assaybiological fluids of either animals or humans. Such fluids include, butare not limited to, whole blood, plasma, sera, lymph, bile, urine,spinal fluid, sputum, perspiration and the like as well as stoolsecretions. It is also possible to assay fluid preparations of human oranimal tissue such as skeletal muscle, heart, kidney, lungs, brains,bone marrow, skin and the like.

In the assay of this invention, the ligand to be determined and thecorresponding labeled ligand analog compete for a fixed amount of commonreactant. This reactant which specifically recognizes the ligand andligand analog and reacts to form complexes with them is referred toherein as the receptor.

The method of this invention is practiced with a dry analytical element.The simplest element can be composed of an absorbent carrier material,e.g. a thin sheet of a self-supporting absorbent or bibulous material,such as filter paper or strips, which contains the binder compositiondescribed below and any other desired reagents. Alternatively, thereagents needed for an assay can be added to the element at the time ofthe assay. The element can be divided into two or more discrete zoneswith different reagents incorporated into individual zones of thecarrier material. Such elements are known in the art as test strips,diagnostic elements, dip sticks, diagnostic agents and the like.

Useful absorbent carrier materials are water insoluble and maintaintheir structural integrity when exposed to water or biological fluidssuch as whole blood or serum. Useful elements can be prepared frompaper, porous particulate structures, porous polymeric films, cellulose,glass fibers, woven and nonwoven fabrics (synthetic and nonsynthetic)and the like. Useful materials and procedures for making such elementsare well known in the art as exemplified in U.S. Pat. Nos. 3,092,465(issued Jun. 4, 1963 to Adams et al), 3,802,842 (issued Apr. 9, 1974 toLange et al), 3,915,647 (issued Oct. 28, 1975 to Wright), 3,917,453(issued Nov. 4, 1975 to Milligan et al), 3,936,357 (issued Feb. 3, 1976to Milligan et al), 4,248,829 (issued Feb. 3, 1981 to Kitajima et al),4,255,384 (issued Mar. 10, 1981 Kitajima et al), and 4,270,920 (issuedJun. 2, 1981 to Kondo et al), and 4,312,834 (issued Jan. 26, 1982 toVogel et al).

Preferably, the absorbent carrier material of the dry analytical elementof this invention is a porous spreading zone. This zone can beself-supporting (i.e. composed of a material rigid enough to maintainits integrity), but preferably it is carried on a separate support. Sucha support can be any suitable dimensionally stable, and preferably,nonporous and transparent (i.e. radiation transmissive) material whichtransmits electromagnetic radiation of a wavelength between about 200and about 900 nm. A support of choice for a particular element should becompatible with the intended mode of detection (fluorescence,transmission or reflectance spectroscopy). Useful supports can beprepared from paper, metal foils, films of polystyrene, polyesters [e.g.poly(ethylene terephthalate)], polycarbonates, cellulose esters (e.g.cellulose acetate), etc.

The porous spreading zone of the element can be prepared from anysuitable fibrous or non-fibrous material or mixtures of either or both,as described in U.S. Pat. Nos. 4,292,272 (issued Sep. 29, 1981 toKitajima et al), 3,992,158 (noted above) 4,258,001 (issued Mar. 24, 1981to Pierce et al) and 4,430,436 (issued Feb. 7, 1984 to Koyama et al) andJapanese Patent Publication 57(1982)-101760 (published Jun. 24, 1982).It is desirable that the spreading zone be isotropically porous, meaningthat the porosity is the same in each direction in the zone as caused byinterconnected spaces or pores between particles, fibers, polymericstrands, etc.

The elements can have two or more discrete zones, either in the samelayer or superimposed. At least one of which is preferably a porousspreading zone. The other zones can be reagent zones, registrationzones, additional spreading zones, radiation-blocking or filter zones,subbing zones, barrier zones, etc. as those zones are known in the art.The zones are generally in fluid contact with each other, meaning thatfluids, reagents and reaction products (e.g. color dyes) can pass or betransported between regions of adjacent zones. In other words, when theelement is contacted with fluid, reagents become mixed and can readilymove within the element. Preferably, each zone is a separately coatedlayer, although two or more zones can be separate regions in a singlelayer of the element.

The peroxidase-labeled ligand analog can be present in any zone or layeras long as it is uniformly distributed in the water-soluble bindercomposition described below. In a preferred embodiment, thewater-soluble binder forms a distinct zone or layer in the element. Morepreferably, this zone or layer is adjacent to the porous spreadinglayer, although the two layers can be separated by a subbing orintermediate layer if desired.

The practice of this invention allows one to determine the amount ofunknown ligand in a liquid sample. The ligand can be any immunologicallyreactive compound including, e.g. antigens, haptens, antibodies, toxins,hormones, therapeutic drugs, natural or synthetic steroids, proteins andother species which will complex specifically with a correspondingreceptor. This invention is particularly useful for the determination oftherapeutic drugs, e.g. digoxin.

The ligand analog useful in this invention is formed using any suitabletechnique known to one skilled in the art. Generally, they are preparedby covalently binding the peroxidase label to the ligand molecule whichmay be modified in any suitable way to achieve the binding.

The peroxidase-labeled ligand analog is uniformly distributed in awater-soluble binder composition comprising poly(vinyl alcohol) in anamount of at least about 50% and up to 100%, by weight, of the bindercomposition. Preferably, at least 80%, by weight, of the composition ispoly(vinyl alcohol). The remainder of the composition is preferably oneor more suitable synthetic or natural binder materials which are presentin amounts that do not adversely affect the water-solubility of thecomposition. Examples of useful binder materials which can be used incombination with poly(vinyl alcohol) include gelatin, polyacrylic acid,poly(acrylamide-co-N-vinyl-2-pyrrolidone) (50:50 weight ratio) andsimilar copolymers, poly(N-vinyl-2-pyrrolidone), polyacrylamide, waterabsorbent starch-containing polymers such as those described in U.S.Pat. No. 3,935,099 (issued Jan. 27, 1976 to Weaver et al), and similarmaterials.

The coverage of the water-soluble binder composition in the element isat least about 0.1, and preferably from about 0.5 to about 5, g/m².

The method of this invention is carried out in the presence of areceptor for the ligand to be determined. For example, if the ligand isan antigen, the receptor is the corresponding antibody. The receptorsare generally commercially available, or they can be readily preparedusing known techniques and starting materials. Generally, theappropriate receptors, e.g. antibodies, are produced by inoculating asuitable animal with the ligand to produce antibodies according to anappropriate protocol, and removing the generated antibodies from theanimal. The receptor can be added to the element prior to orsubstantially simultaneously with the test sample.

Alternatively and preferably, the receptor is immobilized within theelement prior to the assay, e.g. during manufacture. For example, it canbe immobilized within the absorbent carrier material. More particularly,it can be immobilized within the porous spreading zone on a carriermaterial, such as glass or polymeric beads or other particles, resins,fibers and the like. One useful carrier material is a microorganism,such as Staphylococcus aureus. Alternatively, the porous spreading zonecomponents, e.g. beads, can serve as the carrier material for thereceptor.

The method of this invention is carried out in such a manner that eithercomplexed or uncomplexed ligand analog is measured. In a preferredembodiment, the method is carried out so that the complex formed betweenthe receptor and labeled ligand is determined. This complex can bedetermined in any of a number of ways. For example, the complex can bedetermined by a competitive radiometric immunoassay. Alternatively andpreferably, the complex is determined using an interactive compositionwhich provides a spectrophotometric signal in the presence of asubstrate for peroxidase. This composition comprises one or morereagents which can react to produce hydrogen peroxide which in turn canreact with a dye precursor in the presence of peroxidase to produce adetectable dye. The interactive composition can be added to the elementat the time of the assay, with or separate from the test sample.Preferably, it is incorporated in the element during manufacture. Whenso incorporated, the individual components of the composition can belocated in one or more zones of the element. Alternatively, some of thereagents of the interactive composition can be incorporated in theelement while others are added at the time of the assay. During theassay, all of the reagents are mixed and interact in the desired manner.The amounts of each component of the interactive composition to be usedin the assay can be readily determined by one skilled in the art.

Useful dye precursors which can be converted into detectable dyes in thepresence of hydrogen peroxide and peroxidase include various leuco dyessuch as imidazole derivatives described, for example, in U.S. Pat. No.4,089,747 (issued May 16, 1978 to Bruschi), E.P. Application 122,641(published Oct. 24, 1984) and Japanese Patent Publications58(1983)-045,557 58(1983)-068009 and 59(1984)-193353, andtriarylmethanes described, for example, in U.S. Ser. No. 612,509, filedMay 21, 1984 by Babb et al.

In a preferred embodiment, the interactive composition comprisesα-glycerol phosphate oxidase and a triarylimidazole leuco dye. Thiscomposition can be used for the determination of digoxin.

The element can have a number of other useful but optional components inone or more zones, including surfactants, buffers, hardeners,antioxidants, solvents, and others known in the art. The amounts ofthese materials are also within the skill of a worker in the art.

In a preferred embodiment, the element contains a phenol or anilineelectron transfer agent which increases the reaction rate of peroxidase.Useful phenol and aniline electron transfer agents include4-hydroxyacetanilide and others described in copending and commonlyassigned U.S. Ser. No. 884,329 filed on even date herewith by McClune,and entitled USE OF PHENOLS AND ANILINES TO INCREASE THE RATE OFPEROXIDASE CATALYZED OXIDATION OF LEUCO DYES.

The amounts of peroxidase-labeled ligand analog and receptor used in thepractice of this invention are readily determined by one skilled in theart. Generally, the ligand analog is present in the element in an amountof at least about 10⁻⁶, and preferably from about 10⁻⁵ to about 10⁻²,g/m². The receptor (whether incorporated in the element or added duringthe assay) is generally used in an amount which provides at least about10⁻⁷, and preferably from about 10⁻⁶ to about 10⁻², g/m². These amountsrefers to the receptor alone and not to the receptor immobilized on acarrier. The receptor is generally immobilized on a carrier in the ratioof at least 1 part of receptor to 10⁶ parts of carrier, by weight.

A variety of different elements, depending on the method of assay, canbe prepared in accordance with the present invention. Elements can beconfigured in a variety of forms, including elongated tapes of anydesired width, sheets, slides or chips.

The method of this invention can be manual or automated. In general,ligand determination is made by taking the element from a supply roll,chip packet or other source and physically contacting it with a sample(e.g. 1-200 μl) of the liquid to be tested so that the sample, ligandanalog, receptor and reagents within the element interact. Such contactcan be accomplished in any suitable manner, e.g. dipping or immersingthe element into the sample or, preferably, by spotting the element byhand or machine with a drop of the sample with a suitable dispensingmeans.

After sample application, the element is exposed to any conditioning,such as incubation, heating or the like, that may be desirable toquicken or otherwise facilitate obtaining any test result.

Once the receptor has complexed with ligand and ligand analog, anysuitable separation technique can be used to vertically or horizontallyseparate bound (or complexed) ligand analog from unbound (oruncomplexed) ligand analog.

In one embodiment, contact of the sample can be accomplished in such amanner that complexation of receptor and ligand and substantialhorizontal separation of uncomplexed and complexed ligand occur duringsample introduction. This contact can be carried out by hand or with amachine using a pipette or other suitable dispensing means to dispensethe test sample. The sample of liquid can be applied to the element in anumber of ways to effect horizontal separation. For example, arelatively large liquid sample (e.g. up to 100 μl) can be applied slowly(e.g. over at least about 5 seconds) in a continuous manner using asuitable dispensing means. Alternatively, the sample can be applied insmall portions, e.g. as a series of two or more droplets (e.g. 0.1 to 1μl) over a period of time (e.g. over at least about 5 seconds).

In another embodiment, horizontal or vertical separation can beaccomplished by slowly adding a wash fluid after the liquid sample hasbeen applied to the element. This wash causes uncomplexed materials tomove away from the complexed materials.

The amount of ligand in the test sample is then determined by passingthe element through suitable apparatus for detecting the receptor-ligandanalog complex directly or a detectable species formed as a result ofthe reaction of peroxidase and substrate (e.g. change in reflection ortransmission density or fluorescence). Alternatively, the uncomplexedligand analog can be determined in a suitable manner.

In the embodiments noted above involving horizontal separation, thecomplexed ligand analog is measured in a finite area in the center ofthe contacted area. The amount of the ligand in the test sample isinversely proportional to the amount of ligand analog measured in thatfinite area. Generally, ligand analog measurement is carried out fromabout 5 to about 500 seconds after the test sample has been applied tothe element.

In the following examples illustrating the practice of this invention,the materials used were obtained as follows: SURFACTANT 10 G surfactantfrom Olin Corporation (Stamford, Conn., U.S.A.), ZONYL FSN fluorocarbonsurfactant from DuPont (Wilmington, Del., U.S.A.), and the remaindereither from Eastman Kodak Company (Rochester, N.Y., U.S.A.) or preparedusing standard procedures and readily available starting materials.

As used in the context of this disclosure and the claims, I.U.represents the International Unit for enzyme activity defined as oneI.U. being the amount of enzyme activity required to catalyze theconversion of 1 micromole of substrate per minute under standard pH andtemperature conditions for the enzyme.

EXAMPLES 1-3: COMPARATIVE EXAMPLE

These examples demonstrate the improved stability of peroxidase observedwith the elements of the present invention as compared to an elementoutside the scope of this invention.

Three elements of this invention were prepared having a format like thatshown in Example 4 below except that the antibodies on S. aureus wereomitted in Examples 1 and 2, and Examples 2 and 3 contained a bindercomposition comprising poly(vinyl alcohol) (0.05-5 g/m²) and gelatin(0.05-5 g/m²) with 50 percent, by weight, of the binder compositionbeing poly(vinyl alcohol).

Control elements were similarly prepared except that the bindercomposition was composed solely of unhardened gelatin (Control A) orpoly-(acrylamide-co-N-vinyl-2-pyrrolidone) (50:50 weight ratio) (ControlB).

The stability of the peroxidase in the ligand analog in each element wasevaluated in the following manner.

A 10 μl sample of fluid containing 100 mmole of α-glycerol phosphate wasapplied to elements which had been incubated, some at 0° C. in thefreezer and others at 22° C./50% relative humidity. Then the elementswere incubated at 37° C. for 5 minutes and the rate of dye formation wasdetermined in the most linear region of a plot of reflection density vs.time (usually after 1-3 minutes of incubation). The percent peroxidaseactivity retained was calculated by dividing the rate of dye formationobserved for the element kept at 22° C./50% relative humidity by thatobserved for the freezer element.

The results of these tests are shown in Table I below. It is apparentfrom these data that the elements of the present invention havesignificantly improved peroxidase stability (i.e. retained peroxidaseactivity). Example 1 shows the most improvement since high peroxidaseactivity is retained for up to 3 weeks. However, Examples 2 and 3exhibited improved keeping over the Control elements (at least 60%retained activity after 1 week keeping in an open container). BothControl elements had lost a significant amount of peroxidase activityeven after the one week keeping period.

                  TABLE I                                                         ______________________________________                                               Peroxidase Activity Retained                                                    1 Week    1 Week     3 Weeks 3 Weeks                                 Element  (Open)*   (Closed)** (Open)  (Closed)                                ______________________________________                                        Example 1                                                                              about 100%                                                                              97%        about 100%                                                                            98%                                     Example 2                                                                              72%       78%        44%     54%                                     Example 3                                                                              69%       76%        44%     54%                                     Control A                                                                              56%       63%        27%     39%                                     Control B                                                                              48%       59%        19%     34%                                     ______________________________________                                         *Keeping at 22° C. and 50% relative humidity.                          **Keeping at 22° C. and 50% relative humidity in an enclosed           environment.                                                             

EXAMPLE 4: DETERMINATION OF DIGOXIN

This example illustrates the practice of this invention for thedetermination of digoxin. An element of the present invention wasprepared having the following format and components:

    ______________________________________                                        Spreading                                                                             Poly(vinyltoluene- --co- -p- .sub.- t-                                                          50-300    g/m.sup.2                                 Layer   butylstyrene- --co-meth-                                                      acrylic acid) beads                                                           Poly(methylacrylate- --co-2-                                                                    1-10      g/m.sup.2                                         acrylamido-2-methylpro-                                                       pane sulfonic acid- --co-2-                                                   acetoacetoxyethyl meth-                                                       acrylate) adhesive                                                            2-(3,5-Dimethoxy-4-hydroxy                                                                      0.01-1    g/m.sup.2                                         phenyl)-4,5-bis(4-di-                                                         methylaminophenyl)imid-                                                       azole leuco dye                                                               Staphylococcus aureus                                                                           0.2-5     g/m.sup.2                                         coated with digoxin                                                           antibodies                                                                    SURFACTANT 10G    0.1-10    g/m.sup.2                                         surfactant                                                                    Dimedone antioxidant                                                                            0.005-0.5 g/m.sup.2                                 Water   Poly(vinyl alcohol)                                                                             0.1-10    g/m.sup.2                                 Soluble ZONYL FSN surfactant                                                                            0.01-1    g/m.sup.2                                 Layer   Potassium phosphate                                                                             0.01-1    g/m.sup.2                                         buffer (pH 7)                                                                 Digoxin-peroxidase                                                                              10.sup.-6 -10.sup.-4                                                                    g/m.sup.2                                         conjugate                                                             Reagent Gelatin (hardened)                                                                              1-100     g/m.sup.2                                 Layer   SURFACTANT 10G    0.02-2    g/m.sup.2                                         surfactant                                                                    Potassium phosphate                                                                             0.05-5    g/m.sup.2                                         bufer (pH 7)                                                                  α-Glycerol phosphate                                                                      200-20,000                                                                              I.U./m.sup.2                                      oxidase                                                                       4-Hydroxyacetanilide                                                                            0.01-1    g/m.sup.2                                 Poly(ethylene terephthalate)                                                  Support                                                                       ______________________________________                                    

Digoxin was determined using this element in the following manner. Aseries of test samples containing various amounts of the ligand,digoxin, were prepared in a buffered solution (pH 7). A 10 μl sample ofeach test sample was applied to the element prior to incubation forabout 5 minutes at 37° C. At this time, a 10 μl sample of a wash fluidcontaining 100 mmole of α-glycerol phosphate was applied to the elementover the area of the spreading layer contacted with the test sample towash uncomplexed ligand analog horizontally away from complexed ligandanalog, and to initiate the enzymatic reactions which produce adetectable dye. Complexed ligand analog was then determined bymonitoring reflection densities at 670 nm in the center of the spottedarea using a standard reflectometer. The rate of change in dye densitywas calculated from measurements taken between 60 and 120 seconds afterapplication of the second fluid. The Clapper-Williams transform (J.Optical Soc. Am., 43, 595, 1953) was used to determine transmissiondensity values from reflectance density values. The concentration ofdigoxin in the test fluid was observed to be inversely related to therate of dye formation.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

I claim:
 1. An analytical element comprising an absorbent carriermaterial, said element divided into two or more discrete zones, andcontaining in one of said zones a peroxidase-labeled ligand analog foran immunologically reactive ligand uniformly distributed in awater-soluble binder composition comprising at least about 50 percent,by weight, of poly(vinyl alcohol).
 2. The element of claim 1 furthercomprising an interactive composition which is capable of reacting withsaid ligand analog to provide a spectrophotometric signal in thepresence of a substrate for peroxidase.
 3. The element of claim 2wherein said interactive composition comprises α-glycerol phosphateoxidase and a triarylimidazole leuco dye.
 4. The element of claim 2further comprising in at least one of said zones an immobilized receptorfor said immunologically reactive ligand.
 5. The element of claim 1wherein said binder composition comprises an additional binder materialselected from the group consisting of gelatin, polyacrylic acid,acrylamide-N-vinyl-2-pyrrolidone copolymers, polyacrylamide,poly(N-vinyl-2-pyrrolidone) and water-absorbent starch-containingpolymers.
 6. The element of claim 1 wherein poly(vinyl alcohol) ispresent in the binder composition in an amount of at least about 80percent, by weight.
 7. A multilayer analytical element comprising anonporous support having thereon, in order,a registration layer, awater-soluble layer containing a peroxidase-labeled ligand analog for animmuno-logically reactive ligand uniformly distributed in awater-soluble binder composition comprising at least about 50 percent,by weight, of poly(vinyl alcohol), and a porous spreading layer, saidelement further comprising an interactive composition which is capableof interacting with said ligand analog to provide a spectrophotometricsignal in the presence of a substrate for peroxidase.
 8. The element ofclaim 7 wherein said ligand analog is peroxidase-labeled digoxin.
 9. Theelement of claim 7 wherein said interactive composition comprisesα-glycerol phosphate oxidase and a triarylimidazole leuco dye.
 10. Theelement of claim 7 further comprising an immobilized receptor for saidimmunologically reactive ligand in said spreading layer.
 11. The elementof claim 7 wherein said ligand analog is present in a coverage of atleast about 10⁻⁶ g/m².
 12. A method for the determination of animmunologically reactive ligand comprising the steps of:A. in thepresence of a receptor for said ligand, contacting a sample of a liquidsuspected of containing said ligand with an analytical elementcomprising an absorbent carrier material, said element divided into twoor more discrete zones, and containing in one of said zones aperoxidase-labeled ligand analog for said ligand uniformly distributedin a water-soluble binder composition comprising at least about 50percent, by weight, of poly(vinyl alcohol), said contacting carried outin such a manner as to form a complex of receptor and ligand analog, andB. determining the amount of said ligand as a result of the presence ofcomplexed or uncomplexed ligand analog.
 13. The method of claim 12wherein said receptor for said ligand is immobilized in one of saidzones of said element.
 14. The method of claim 12 wherein said liquidsample is contacted with said element in such a manner that saidreceptor-ligand analog complex is immobilized in said element andhorizontal separation of uncomplexed ligand analog from immobilizedcomplex is effected.
 15. The method of claim 14 wherein said separationis accomplished with a wash step subsequent to said contacting step. 16.The method of claim 12 wherein said element comprises a nonporoussupport having thereon, in order,a registration layer, a layercontaining said ligand analog uniformly distributed in a water-solublebinder composition comprising at least about 50 percent, by weight, ofpoly(vinyl alcohol), and a porous spreading layer as said absorbentcarrier material.
 17. The method of claim 12 for the determination ofdigoxin in a biological fluid wherein said ligand analog isperoxidase-labeled digoxin.
 18. The method of claim 12 wherein saidcomplexed ligand analog is measured.
 19. A multilayer analytical elementcomprising a nonporous support having thereon, in order,a porousspreading layer, and a water-soluble layer containing aperoxidase-labeled ligand analog for an immunologically reactive liganduniformly distributed in a water-soluble binder composition comprisingat least about 50 percent, by weight, of poly(vinyl alcohol).